ARA7(Q69L) expression in transgenic Arabidopsis cells induces the formation of enlarged multivesicular bodies.
ARA7(Q69L) expression in transgenic Arabidopsis cells induces the formation of enlarged multivesicular bodies.
复制标题
DOI：
10.1093/jxb/ert125
复制
发表时间：
2013-07
影响因子：
6.9
通讯作者：
Jiang L
中科院分区：
文献类型：
--
作者：
Jia T;Gao C;Cui Y;Wang J;Ding Y;Cai Y;Ueda T;Nakano A;Jiang L
拟南芥ARA 7（AtRabF 2b）是植物Rab 5小GTP酶家族中的一员，在植物细胞中定位于液泡前区室（pre-vacuolar compartments，PVC），即多泡体（multiple-esicular bodies，MVB）。ARA 7的组成型活性GTP结合突变体ARA 7（Q69 L）的过表达诱导形成大的环状结构（直径1-2 μm）。为了更好地理解这些ARA 7（Q69 L）诱导的环状结构的生物学，产生了在热休克诱导型启动子控制下表达用绿色荧光蛋白（GFP）标记的ARA 7（Q69 L）的转基因拟南芥细胞系。在这些转基因细胞中，热休克诱导4小时后形成了坚固的环状结构。瞬时共表达，共聚焦成像，和免疫金电子显微镜（免疫金-EM）实验表明，这些GFP-ARA 7（Q69 L）标记的环状结构是不同的高尔基体和trans-Golgi网络，但标记的抗体对MVB标记蛋白。此外，活细胞成像和详细的EM分析显示GFP-ARA 7（Q69 L）诱导的球形结构起源于MVB的同型融合。总之，证明了GFP-ARA 7（Q69 L）表达是研究植物细胞中PVC/MVB介导的蛋白运输和液泡降解的有效工具。
Arabidopsis thaliana ARA7 (AtRabF2b), a member of the plant Rab5 small GTPases functioning in the vacuolar transport pathway, localizes to pre-vacuolar compartments (PVCs), known as multivesicular bodies (MVBs) in plant cells. Overexpression of the constitutively active GTP-bound mutant of ARA7, ARA7(Q69L), induces the formation of large ring-like structures (1–2 µm in diameter). To better understand the biology of these ARA7(Q69L)-induced ring-like structures, transgenic Arabidopsis cell lines expressing ARA7(Q69L) tagged with green fluorescent protein (GFP) under the control of a heat shock-inducible promoter were generated. In these transgenic cells, robust ring-like structures were formed after 4 h of heat shock induction. Transient co-expression, confocal imaging, and immunogold electron microscopy (immunogold-EM) experiments demonstrated that these GFP–ARA7(Q69L)-labelled ring-like structures were distinct from the Golgi apparatus and trans-Golgi network, but were labelled with an antibody against an MVB marker protein. In addition, live cell imaging and detailed EM analysis showed that the GFP–ARA7(Q69L)-induced spherical structures originated from the homotypic fusion of MVBs. In summary, it was demonstrated that GFP–ARA7(Q69L) expression is an efficient tool for studying PVC/MVB-mediated protein trafficking and vacuolar degradation in plant cells.
登录
查看更多内容
影响因子：
64.5
作者：
Geldner, N;Anders, N;Jürgens, G
通讯作者：
Jürgens, G
影响因子：
4.9
作者：
Lee, GJ;Sohn, EJ;Hwang, I
通讯作者：
Hwang, I
影响因子：
56.9
作者：
Kim, Sun A.;Punshon, Tracy;Guerinot, Mary Lou
通讯作者：
Guerinot, Mary Lou
影响因子：
11.6
作者：
Gao, Caiji;Yu, Christine K. Y.;Jiang, Liwen
通讯作者：
Jiang, Liwen
影响因子：
4
作者：
Kotzer, AM;Brandizzi, F;Hawes, C
通讯作者：
Hawes, C